XII. Gene Regulation.

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2 - Overview:All cells in an organism contain the same genetic information; the key to tissue specialization is gene regulation – reading some genes in some cells and other genes in other cells.

3 Also, organisms can respond to their environment at a genetic level, so there must be a way for the environment to stimulate or repress the action of certain genes.

4 And changes occur through time, creating developmental changesAnd changes occur through time, creating developmental changes. We will look at how gene expression is regulated in these cases.

5 - Overview:Some Terminology:some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.

6 - Overview:Some Terminology:some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present.

7 - Overview:Some Terminology:some enzymatic genes are only turned on if the substrate is present; this is an inducible system and the substrate is the inducer. Obviously, this is highly adaptive, as the cell saves energy by only producing the enzyme when it is needed.some enzymes are on all the time, and are only turned off if a compound (often the product of the metabolic process they are involved with) is present. This is a repressible system, and the compound is the repressor. This is also adaptive, and the cell saves on enzymes if the product is already present.Constitutive genes are on all the time.

9 XII. Gene RegulationA. The lac Operon in E. coliWhen lactose is present, E. coli produce three enzymes involved in lactose metabolism. Lactose is broken into glucose and galactose, and galactose is modified into glucose, too. Glucose is then metabolized in aerobic respiration pathways to harvest energy (ATP). When lactose is absent, E. coli does not make these enzymes and saves energy and amino acids. How do they KNOW? : )

10 XII. Gene RegulationA. The lac Operon in E. coliAs you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.

11 XII. Gene RegulationA. The lac Operon in E. coliAs you remember, an “operon” was a region of genes that are regulated as a unit – it typically encodes > 1 protein involved in a particular metabolic pathway.

15 XII. Gene RegulationThe lac Operon in E. coli1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription.Repressor GeneOperatorRepressorRNA Poly

16 XII. Gene RegulationThe lac Operon in E. coli1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription.LACTOSE

17 The binding of lactose changes the shape of the repressor (allosteric reaction) and it can’t bind to the operator.XII. Gene RegulationThe lac Operon in E. coli1960 – Jacob and Monod proposed that this was an inducible system because the presence of the substrate INDUCES transcription.LACTOSE

21 XII. Gene RegulationThe lac Operon in E. coliMutant analyses confirmed these results:Curiously, there are only about 10 repressor molecules in each cell and they were not actually isolated and identified for 6 years (Gilbert).

22 XII. Gene RegulationThe lac Operon in E. coliBut it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW?

23 XII. Gene RegulationThe lac Operon in E. coliBut it is even more complicated… if glucose AND lactose are present, the operon is OFF. This is adaptive, because it’s glucose the cell needs. If glucose is present, there is no need to break lactose down to get it. BUT HOW?This involves a repressible pathway.

24 XII. Gene Regulation The lac Operon in E. coliWithin the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter., lactose present

25 XII. Gene Regulation The lac Operon in E. coliWithin the promoter, there is a binding site for Catabolic Activating Protein – basically a “transcription factor”. CAP needs to bind in order for the RNA Polymerase to bind. Cyclic-AMP activates CAP, causing an allosteric reaction so it can bind the promoter. So, the binding of CAP stimulates transcription., lactose present

26 XII. Gene Regulation The lac Operon in E. coliWhen Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off., lactose present

28 XII. Gene RegulationThe lac Operon in E. coliWhen Glucose is present, the concentration of c-AMP declines, it does not bind to CAP, and CAP does not bind to the Promoter; so the RNA Poly does not bind either and the genes are off.So, the lac operon is regulated first by the presence/absence of glucose; the needed nutrient…and then by the presence of lactose, which could be metabolized to produce glucose if necessary.

30 XII. Gene RegulationThe lac Operon in E. coliB. The trp Operon in E. coliTryptophan is an amino acid that can be synthesized by tryptophan synthetase. This gene and its partners are only ON if tryptophan is absent. The presence of tryptophan represses the production of these enzymes (repressible system).

32 B. The trp Operon in E. coliACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…

33 B. The trp Operon in E. coliACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…

34 B. The trp Operon in E. coliACTUALLY, TRANSCRIPTION ALWAYS PROCEEDS A LITTLE BIT…UP TO THE REGION CALLED THE “ATTENUATOR”…

35 B. The trp Operon in E. coliTwo hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription.

36 B. The trp Operon in E. coliTwo hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription.Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence.

37 B. The trp Operon in E. coliTwo hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription.Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence.If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms…

38 B. The trp Operon in E. coliTwo hairpin loops can form in the m-RNA; the 3-4 loop causes termination of transcription.Because translation occurs as soon as m-RNA is produced, ribosomes jump on and begin to read the strand… there are two trp codons at the beginning of the sequence.If trp is present, the ribosome zooms along (incorporating trp) and it occupies the 2 region… region 3 is free to bind with 4 and the termination loop forms…If low trp, then ribosome stalls; region 3 bind to 2, no termination loop forms, and transcription of the genes proceeds…Translation of the genes begins at start codons downstream…

41 XII. Gene RegulationThe lac Operon in E. coliB. The trp Operon in E. coliC. Regulation in Eukaryotes- higher levels of packaging, intron-exon structure, and the need for tissue specialization makes regulation in eukaryotes far more complex that responding to environmental cues.Histone Regulation- Core DNA, bound to histones, is OFF. Only “linker DNA”, between histones, is even accessible to RNA polymerases. So, binding DNA to histones is the first way to shut it off.

45 C. Regulation in EukaryotesHistone RegulationMethylationPromoters- Several consensus sequences (TATA, CAAT, GGGCGG) appear in combination in nearly all promoters and are required for basal levels of transcription

46 C. Regulation in EukaryotesHistone RegulationMethylationPromotersEnhancers/SilencersCis-acting elements on the same chromosome, which regulate a neighboring gene.They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or after

47 C. Regulation in EukaryotesHistone RegulationMethylationPromotersEnhancers/SilencersCis-acting elements on the same chromosome, which regulate a neighboring gene.They are somewhat like operators, in that they are binding sites for transcription factors that can “up” or “down” regulate transcription. However, they function ANYWHERE near the gene: before, within, or afterThey are not gene specific – they will enhance their neighborSilencers tend to reduce binding of the polymerase to the promoter.

48 C. Regulation in EukaryotesHistone RegulationMethylationPromotersEnhancers/SilencersThese are the transcription factors that bind to enhancer and silencer regions of the human metallothionien IIA gene promoter region!!- What does having all these modifiers allow for?

49 C. Regulation in EukaryotesEnhancers/SilencersTranscription Factors- These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.

50 C. Regulation in EukaryotesEnhancers/SilencersTranscription Factors- These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.HTH = “helix-turn-helix”One class of important HTH TF’s contain specific sequences of AA’s called a homeodomain. This is encoded by a 180 bp region in it’s gene called a homeobox. These homeotic genes/proteins are conserved across all eukaryotes and are critical to basic animal development.

51 C. Regulation in EukaryotesEnhancers/SilencersTranscription Factors- These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.“Zinc-Finger”: Zinc binds to two cysteine andtwo histidine AA’s. The sequence between formsA loop or “finger”, and the specific AA sequenceBinds specific DNA sequences…

53 C. Regulation in EukaryotesEnhancers/SilencersTranscription Factors- These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.bZIP=“basic leucine zipper”: leucine AA’s inDifferent chains dimerize and the leucines “zip”The other alpha-helices bind specific DNA sequences

54 C. Regulation in EukaryotesEnhancers/SilencersTranscription Factors- These are the proteins that bind to DNA and influence transcription. They have “binding domains” that bind DNA in particular ways.- Then, the TF’s have other biding sites forProteins (like basal transcription factors) orOther chemicals (like hormones)

55 C. Regulation in Eukaryotes Transcription Factors Alternate Splicing Pathways- Many proteins can be made from the same gene, by splicing the m-RNA differently. Humans have 20-30K genes, but several 100,000 proteins!A calcium regulator in the thyroidA hormone made in the brain

56 C. Regulation in EukaryotesAlternate Splicing Pathways7. Controlling m-RNA stabilityExisting tubulin units interact with a new tubulin strand and translation stalls, releasing RNAse that cleave the m-RNA.So tubulin is only made when free tubulin units are not present.

57 C. Regulation in EukaryotesControlling m-RNA stabilityRNA Silencing- Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression.

58 C. Regulation in EukaryotesControlling m-RNA stabilityRNA Silencing- Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression.- si-RNA: Initially they are found as ds-RNA(probably as the result of hairpin); theyare cut by the ‘dicer’ protein into 21 basesequences (short-interfering RNA’s ) thatbind a protein and, as ss-RNA, bind m-RNAand the protein cleaves the m-RNA.

59 C. Regulation in EukaryotesControlling m-RNA stabilityRNA Silencing- Short pieces of RNA can bind to DNA in the nucleus or m-RNA in the cytoplasm and regulate gene expression.- si-RNA: Initially they are found as ds-RNA(probably as the result of hairpin); theyare cut by the ‘dicer’ protein into 21 basesequences (short-interfering RNA’s ) thatbind a protein and, as ss-RNA, bind m-RNAand the protein cleaves the m-RNA.- miRNA (microRNA): quite similar, but asss-RNA they bind m-RNA and just stoptranslation. They are involved in developmentalregulation – many bind m-RNA for transcription factors.They also can bind DNA and induce methylation in promoters, turninggenes off. This is all RNA silencing……

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